Macroautophagy (autophagy) is an important mechanism for targeting cellular components including proteins, protein aggregates and organelles for degradation in lysosomes. This catabolic, cellular self-digestion process is induced in response to starvation or stress, causing the formation of double membrane vesicles called autophagosomes that engulf proteins and organelles. Autophagosomes then fuse with lysosomes where the autophagosome and their cargo are degraded. This lysosome-mediated cellular self-digestion serves to recycle intracellular nutrients to sustain cell metabolism during starvation and to eliminate damaged proteins and organelles that accumulate during stress. Although elimination of individual proteins occurs by the ubiquitin-mediated proteasome degradation pathway, the autophagy pathway can eliminate protein aggregates and organelles. Thus, autophagy complements and overlaps with proteasome function to prevent the accumulation of damaged cellular components during starvation and stress. Through these functions, autophagy is an essential cellular stress response that maintains protein and organelle quality control, protects the genome from damage, and sustains cell and mammalian viability.
Autophagy is thought to be controlled by ATG proteins, initially identified in yeast, for which there are mammalian homologues (Levine, B., and Kroemer, G. (2008), Autophagy in the pathogenesis of disease, Cell 132, 27-42). ATG proteins are comprised of kinases, proteases, and two ubiquitin-like conjugation systems that likely function in concert with a host of unknown cellular proteins to control autophagosome formation, cargo recognition, engulfment, and trafficking to lysosomes.
In mice, autophagy enables survival of neonatal starvation by preventing energy depletion. Mice with targeted autophagy-deficiency (atg5−/− or atg7−/−) in the brain accumulate damaged mitochondria and polyubiquitin-containing protein aggregates, and display neuronal degeneration. Defects in autophagy through liver-specific atg7 deletion in mice similarly results in protein aggregate accumulation, hepatocyte cell death and severe liver injury. These findings support a prosurvival role for autophagy in sustaining cellular metabolism and maintaining protein and organelle quality control by eliminating damaged proteins and organelles that are particularly important during nutrient stress and aging (Levine, B. and Kroemer, G., (2008)).
Autophagy dysfunction is a major contributor to diseases including, but not limited to, neurodegeneration, liver disease, and cancer. Many human neurodegenerative diseases are associated with aberrant mutant and/or polyubiquitinated protein accumulation and excessive neuronal cell death. Neurons of mice with targeted autophagy defects accumulate polyubiquitinated- and p62 containing protein aggregates that result in neurodegeneration. The human liver disease steatohepatitis and a major subset of hepatocellular carcinomas (HCCs) are associated with the formation of p62-containing protein aggregates (Mallory bodies) (Zatloukal, K., et al. (2002), p62 is a common component of cytoplasmic inclusions in protein aggregation diseases, Am. J. Pathol. 160, 255-263). Livers of mice with autophagy defects have p62-containing protein aggregates, excessive cell death, and HCC.
Autophagy is also induced by stress and starvation in tumor cells where it predominantly provides a prosurvival function. Metabolic stress is common and autophagy localizes to metabolically-stressed tumor regions. Autophagy has been identified as an important survival pathway in epithelial tumor cells that enables long-term survival to metabolic stress (Degenhardt, K., et al. (2006), Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis, Cancer Cell 10, 51-64; Jin, S., and White, E. (2007), Role of autophagy in cancer: management of metabolic stress. Autophagy 3, 28-31; Karantza-Wadsworth, V., et al., (2007), Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis, Genes Dev 21, 1621-1635; Mathew, R. et al., (2007a), Role of autophagy in cancer, Nat Rev Cancer 7, 961-967; Mathew, R., et al. (2007b), Autophagy suppresses tumor progression by limiting chromosomal instability, Genes Dev 21, 1367-1381). Tumor cells with defined defects in autophagy accumulate p62-containing protein aggregates, DNA damage, and die in response to stress, whereas those with intact autophagy can survive for weeks utilizing the autophagy survival pathway. Thus, autophagy may be required to prevent tumor cell damage and to maintain metabolism. Tumor cells can exploit this survival function to remain dormant only to reemerge under more favorable conditions.
Paradoxically, autophagy defects through allelic loss of the essential autophagy gene beclin1 or through constitutive activation of the autophagy-suppressing PI-3 kinase/mTOR pathway are common in human tumors. Roughly half of human cancers may have impaired autophagy, either due to constitutive activation of the PI-3 kinase pathway or allelic loss of the essential autophagy gene beclin1, rendering them particularly susceptible to metabolic stress and autophagy inhibition (Jin et al., 2007; Jin, S., and White, E. (2008), Tumor suppression by autophagy through the management of metabolic stress, Autophagy 4, 563-566).
Analogous to a wound-healing response, chronic tumor cell death in response to stress and induction of inflammation and cytokine production may provide a non-cell-autonomous mechanism by which tumorigenesis is promoted in autophagy-defective cells. Autophagy-defective tumor cells also display an elevated DNA damage response, gene amplification and chromosome instability in response to stress, suggesting that autophagy limits genome damage as a cell-autonomous mechanism of tumor suppression. Possible non-mutually exclusive mechanisms by which autophagy may protect the genome include maintenance of metabolism and ATP levels, reduction of oxidative stress, and elimination of damaged protein and organelles.
The importance of autophagy in cellular garbage disposal is clear, as autophagy is the only identified mechanism for the turnover of large cellular structures such as organelles and protein aggregates. How organelles are recognized and directed to autophagosomes for degradation may involve organelle-specific processes such as mitophagy and ER-phagy that may mitigate oxidative stress emanating from dysfunctional organelles. Damaged proteins that accumulate during stress can be refolded, ubiquitinated and degraded by the proteasome pathway, or aggregated and degraded by autophagy. To direct damaged or unfolded proteins to the autophagy pathway, p62 binds to polyubiquitinated proteins forming protein aggregates by oligomerization and to Atg8/LC3 on the autophagosome membrane to target aggregates to autophagosomes for degradation. Protein aggregation may be a protective mechanism to limit cellular exposure to toxic proteins through sequestration, as well as an efficient packaging and delivery mechanism that collects and directs damaged proteins to autophagosomes. Liver-specific autophagy defects in mice cause accumulation of p62 aggregates, elevated oxidative stress and hepatocyte cell death. Thus, without seeking to be bound by any theory or theories of operation, it is believed that the inability to dispose of p62 aggregates through autophagy may be toxic to normal tissues.
The ATG6/Beclin1-Vps34-ATG8/LC3 complex regulates autophagosome formation; LC3 cleavage, lipidation, and membrane translocation are frequently utilized to monitor autophagy induction. The mechanism by which starvation and stress activate autophagy is controlled in part through the PI-3 kinase pathway via the protein kinase mTOR. Growth factor and nutrient availability promote mTOR activation that suppresses autophagy, whereas starvation and mTOR inactivation stimulate autophagy (Klionsky (2007), Nat Rev Mol Cell Biol 8, 931-937). While there are other mechanisms to regulate autophagy, mTOR provides a link between nutrient and growth factor availability, growth control, autophagy, and metabolism.
Autophagy is believed to play an essential role in maintaining protein quality control, while defective autophagy may be involved in the development of diseases including, but not limited to, neurodegeneration, steatohepatitis, and cancer. Therefore, there exists a need for identification of stimulators of autophagy.
Additionally, there exists a need for the identification of inhibitors of the autophagy survival pathway in, for example, cancer cells. Such inhibitors of autophagy could be used in the prevention and/or treatment of cancer.